The present invention is related to the area of chemical synthesis. More specifically, one embodiment of the present invention provides certain photolabile compounds and methods for their use as photocleavable linkers.
The use of a photolabile molecule as a linker to couple peptides to solid supports and to facilitate the subsequent cleavage reaction has received considerable attention during the last two decades. Photolysis offers a mild method of cleavage which complements traditional acidic or basic cleavage techniques. See, e.g., Lloyd-Williams et al. (1993) Tetrahedron 49: 11065-11133. The rapidly growing field of combinatorial organic synthesis (see, e.g., Gallop et al. (1994) J. Med. Chem. 37: 1233-1251; and Gordon et al. (1994) J. Med. Chem. 37: 1385-1401) involving libraries of peptides and small molecules has markedly renewed interest in the use of photolabile linkers for the release of both ligands and tagging molecules.
A phenacyl based linking group (see 1 below) has been described. See Wang, (1976) J. Org. Chem. 41: 3258. ##STR1##
An ortho-nitrobenzyl support (see 2 below) derived from 4-bromomethyl-3-nitrobenzoic acid has been widely employed as a photolabile support for the generation of both peptide acids and amides. See Rich et al. (1975) J. Am. Chem. Soc. 97: 1575-1579 and Hammer et al. (1990) Int. J. Peptide Protein Res. 36: 31-45. ##STR2## Photolabile support 2, though useful, does suffer from several limitations. For example, workers have been unable to obtain high yields of methionine-containing peptides from the support without substantial contamination with methionine sulfoxide. See Rich supra and Hammer supra. One solution has been to employ methionine sulfoxide throughout the peptide assembly and to subsequently reduce back to methionine to avoid any ambiguities associated with partial oxidation (see, Lloyd-Williams et al. (1991) J. Peptide Protein Res. 37: 58-60 and Lloyd-Williams et al. (1993) Tetrahedron 49: 10069-10078), but this clearly detracts from the usefulness of the technique. This support also suffers from unduly slow cleavage kinetics, with typical photolysis times in organic solvents ranging from 12 to 24 hours. Moreover, photolysis of the support generates a reactive and chromogenic nitroso-aldehyde on the support which can trap liberated compounds and may act as an internal light filter to slow the rate of cleavage. See Patchnornik et al. (1970) J. Am. Chem. Soc. 92: 6333-6335.
Pillai and co-workers have described an .alpha.-methyl-ortho-nitrobenzyl support designed to eliminate formation of the nitroso-aldehyde, but they observed inefficient release of peptides longer than five residues due to poor swelling of the resin. See Ajayaghosh et al. (1988) Tetrahedron 44: 6661-6666.
In the course of optimizing the photolithographic synthesis of both peptides (see Fodor et al. (1991) Science 251: 767-773) and oligonucleotides (see Pease et al. (1994) Proc. Natl. Acad. Sci. USA 91: 5022-5026, we had occasion to explore the use of a variety of ortho-benzyl compounds as photolabile protecting groups. See PCT patent publication Nos. WO 90/15070, WO 92/10092, and WO 94/10128; see also U.S. patent application Ser. No. 07/971,181, filed Nov. 2, 1992, and Ser. No. 08/310,510, filed Sep. 22, 1994; Holmes et al. (1994) in Peptides: Chemistry, Structure and Biology (Proceedings of the 13th American Peptide Symposium): Hodges et al. Eds.; ESCOM: Leiden; pp. 110-12, each of these references is incorporated herein by reference for all purposes. Examples of these compounds included the 6-nitroveratryl derived protecting groups, which incorporate two additional alkoxy groups onto the benzene ring. Introduction of an .alpha.-methyl onto the benzylic carbon facilitated the photolytic cleavage with &gt;350 nm UV light and resulted in the formation of a nitroso-ketone.
Photolabile amide protecting groups for C-termini of peptides which rely on the same basic ortho-nitro benzyl chemistry have also been reported. See Henricksen et al. (1993) Int. J. Peptide Protein Res. 41: 169-180; Ramesh et al. (1993) J. Org. Chem. 58: 4599-5605; Pillai (1980) Synthesis 1-26; and Pillai et al. (1979) Tetrahedron Lett. 3409-3412. See also Bellof and Mutter (1985) Chimia 39: 10.
A photocleavable linker should be stable to variety of reagents (e.g., piperidine, TFA, and the like); be rapidly cleaved under mild conditions; and not generate highly reactive byproducts. The present invention provides such linkers.